Ensuring Optimal Medium pH for Human Pluripotent Stem Cells

As we move towards clinical applications using the differentiated derivatives of human pluripotent stem cells (hPSCs), there has been a renewed focus on cell quality. Of particular concern is the genomic stability of hPSCs which have been found to frequently acquire recurrent genetic abnormalities in a non-random and sporadic manner. As such, it is important to routinely assess cell quality and minimize any cell stressors in the culture system. 

Environmental stressors such as reactive oxygen species (ROS), nutrient exhaustion, and build-up of metabolites such as lactic acid have been investigated for their potential contribution to DNA damage, which in turn may increase the likelihood of genetic abnormalities (1, 2). Interestingly, acidification of the extracellular environment has been identified as a key stressor, with pH below 7.0 shown to attenuate glycolytic rates (1), and below 6.9 correlated with significant increases in DNA damage (2). Moreover, reducing medium acidity alone has been shown to reduce apoptosis in culture (3).

hPSCs are best maintained within the physiological pH range, i.e. between 7.2 and 7.4 (4). The preferred way to ensure a healthy extracellular pH, historically, has been to provide cells with fresh media every 24 hours. However, recent advancements in medium buffering enable pH to remain stable for up to 72 hours, even at high cell densities, without the need for a medium change.

Figure 1. mTeSR? Plus Maintains Optimal pH Levels Throughout a Weekend-Free Protocol

The pH of spent medium from hPSCs cultured in mTeSR? Plus is higher than that of hPSCs cultured in mTeSR?1 and other flexible-feeding media at similar cell densities. pH and cell numbers were measured after a 72-hour period without feeding. Range of cell numbers shown represent different densities that would be observed throughout a typical passage. This demonstrates that feeds can be skipped for two days at any time during routine maintenance using mTeSR? Plus while maintaining a pH above 7.0. Note: Cultures were fed double the standard medium volume prior to the 72-hour period without feeds in all media and cell numbers are from one well of a 6-well plate.

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References:

1. Wilmes A et al. (2017) Towards optimisation of induced pluripotent cell culture: Extracellular acidification results in growth arrest of iPSC prior to nutrient exhaustion. Toxicol In Vitro 45(3): 445–454
2. Jacobs K et al. (2016) Higher-density culture in human embryonic stem cells results in DNA damage and genome instability. Stem Cell Reports. 6(3): 330–341
3. Liu W et al. (2018) The suppression of medium acidosis improves the maintenance and differentiation of human pluripotent stem cells at high density in defined cell culture medium. Int J Biol Sci. 14(5): 485–496
4. Ludwig T et al. (2006) Derivation of human embryonic stem cells in defined conditions. Nature Biotechnol. 24(2): 185–7